Letter | Published:

A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds

Naturevolume 565pages9195 (2019) | Download Citation


The molecular pathways that trigger the initiation of embryogenesis after fertilization in flowering plants, and prevent its occurrence without fertilization, are not well understood1. Here we show in rice (Oryza sativa) that BABY BOOM1 (BBM1), a member of the AP2 family2 of transcription factors that is expressed in sperm cells, has a key role in this process. Ectopic expression of BBM1 in the egg cell is sufficient for parthenogenesis, which indicates that a single wild-type gene can bypass the fertilization checkpoint in the female gamete. Zygotic expression of BBM1 is initially specific to the male allele but is subsequently biparental, and this is consistent with its observed auto-activation. Triple knockout of the genes BBM1, BBM2 and BBM3 causes embryo arrest and abortion, which are fully rescued by male-transmitted BBM1. These findings suggest that the requirement for fertilization in embryogenesis is mediated by male-genome transmission of pluripotency factors. When genome editing to substitute mitosis for meiosis (MiMe)3,4 is combined with the expression of BBM1 in the egg cell, clonal progeny can be obtained that retain genome-wide parental heterozygosity. The synthetic asexual-propagation trait is heritable through multiple generations of clones. Hybrid crops provide increased yields that cannot be maintained by their progeny owing to genetic segregation. This work establishes the feasibility of asexual reproduction in crops, and could enable the maintenance of hybrids clonally through seed propagation5,6.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data availability

Whole-genome DNA sequencing data for S-Apo line 1 mother plant, the four progeny clones from two generations, and the Kitaake wild-type control are available from National Center for Biotechnology Information (NCBI) BioProject number PRJNA496208. RNA sequencing data from previously published datasets11,15 are available from the NCBI Short Read Archive as Project SRP119200 and from the NCBI Gene Expression Omnibus under accession number GSE50777.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Palovaara, J., de Zeeuw, T. & Weijers, D. Tissue and organ initiation in the plant embryo: a first time for everything. Annu. Rev. Cell Dev. Biol. 32, 47–75 (2016).

  2. 2.

    Horstman, A., Willemsen, V., Boutilier, K. & Heidstra, R. AINTEGUMENTA-LIKE proteins: hubs in a plethora of networks. Trends Plant Sci. 19, 146–157 (2014).

  3. 3.

    d’Erfurth, I. et al. Turning meiosis into mitosis. PLoS Biol. 7, e1000124 (2009).

  4. 4.

    Mieulet, D. et al. Turning rice meiosis into mitosis. Cell Res. 26, 1242–1254 (2016).

  5. 5.

    Sailer, C., Schmid, B. & Grossniklaus, U. Apomixis allows the transgenerational fixation of phenotypes in hybrid plants. Curr. Biol. 26, 331–337 (2016).

  6. 6.

    Vielle Calzada, J.-P., Crane, C. F. & Stelly, D. M. Apomixis—the asexual revolution. Science 274, 1322–1323 (1996).

  7. 7.

    Lee, M. T., Bonneau, A. R. & Giraldez, A. J. Zygotic genome activation during the maternal-to-zygotic transition. Annu. Rev. Cell Dev. Biol. 30, 581–613 (2014).

  8. 8.

    Nodine, M. D. & Bartel, D. P. Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos. Nature 482, 94–97 (2012).

  9. 9.

    Autran, D. et al. Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis. Cell 145, 707–719 (2011).

  10. 10.

    Del Toro-De León, G., García-Aguilar, M. & Gillmor, C. S. Non-equivalent contributions of maternal and paternal genomes to early plant embryogenesis. Nature 514, 624–627 (2014).

  11. 11.

    Anderson, S. N. et al. The zygotic transition is initiated in unicellular plant zygotes with asymmetric activation of parental genomes. Dev. Cell 43, 349–358.e4, (2017).

  12. 12.

    Kim, S., Soltis, P. S., Wall, K. & Soltis, D. E. Phylogeny and domain evolution in the APETALA2-like gene family. Mol. Biol. Evol. 23, 107–120 (2006).

  13. 13.

    Boutilier, K. et al. Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell 14, 1737–1749 (2002).

  14. 14.

    Passarinho, P. et al. BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways. Plant Mol. Biol. 68, 225–237 (2008).

  15. 15.

    Anderson, S. N. et al. Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: evidence for distinct sex-dependent chromatin and epigenetic states before fertilization. Plant J. 76, 729–741 (2013).

  16. 16.

    Conner, J. A., Mookkan, M., Huo, H., Chae, K. & Ozias-Akins, P. A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant. Proc. Natl Acad. Sci. USA 112, 11205–11210 (2015).

  17. 17.

    Conner, J. A., Podio, M. & Ozias-Akins, P. Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes. Plant Reprod. 30, 41–52 (2017).

  18. 18.

    Steffen, J. G., Kang, I. H., Macfarlane, J. & Drews, G. N. Identification of genes expressed in the Arabidopsis female gametophyte. Plant J. 51, 281–292 (2007).

  19. 19.

    Ohnishi, Y., Hoshino, R. & Okamoto, T. Dynamics of male and female chromatin during karyogamy in rice zygotes. Plant Physiol. 165, 1533–1543 (2014).

  20. 20.

    Lowe, K. et al. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. Plant Cell 28, 1998–2015 (2016).

  21. 21.

    Murovec, J. & Bohanec, B. in Plant Breeding (ed. Abdurakhmonov, I. Y.) Ch. 5 (IntechOpen, London, 2012).

  22. 22.

    Cifuentes, M., Rivard, M., Pereira, L., Chelysheva, L. & Mercier, R. Haploid meiosis in Arabidopsis: double-strand breaks are formed and repaired but without synapsis and crossovers. PLoS ONE 8, e72431 (2013).

  23. 23.

    Hand, M. L. & Koltunow, A. M. The genetic control of apomixis: asexual seed formation. Genetics 197, 441–450 (2014).

  24. 24.

    Ozias-Akins, P. & van Dijk, P. J. Mendelian genetics of apomixis in plants. Annu. Rev. Genet. 41, 509–537 (2007).

  25. 25.

    Khush, G. S. Apomixis: exploiting hybrid vigor in rice. (International Rice Research Institute, 1994).

  26. 26.

    Sanger, F., Nicklen, S. & Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74, 5463–5467 (1977).

  27. 27.

    Lafon-Placette, C. & Köhler, C. Endosperm-based postzygotic hybridization barriers: developmental mechanisms and evolutionary drivers. Mol. Ecol. 25, 2620–2629 (2016).

  28. 28.

    Sekine, D. et al. Dissection of two major components of the post-zygotic hybridization barrier in rice endosperm. Plant J. 76, 792–799 (2013).

  29. 29.

    Hua, J. et al. Single-locus heterotic effects and dominance by dominance interactions can adequately explain the genetic basis of heterosis in an elite rice hybrid. Proc. Natl Acad. Sci. USA 100, 2574–2579 (2003).

  30. 30.

    Huang, X. et al. Genomic architecture of heterosis for yield traits in rice. Nature 537, 629–633 (2016).

  31. 31.

    Murashige, T. & Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497 (1962).

  32. 32.

    Khanday, I., Yadav, S. R. & Vijayraghavan, U. Rice LHS1/OsMADS1 controls floret meristem specification by coordinated regulation of transcription factors and hormone signaling pathways. Plant Physiol. 161, 1970–1983 (2013).

  33. 33.

    Aoyama, T. & Chua, N. H. A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 11, 605–612 (1997).

  34. 34.

    Xie, K., Zhang, J. & Yang, Y. Genome-wide prediction of highly specific guide RNA spacers for CRISPR–Cas9-mediated genome editing in model plants and major crops. Mol. Plant 7, 923–926 (2014).

  35. 35.

    Zhou, H., Liu, B., Weeks, D. P., Spalding, M. H. & Yang, B. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res. 42, 10903–10914 (2014).

  36. 36.

    Hiei, Y. & Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protoc. 3, 824–834 (2008).

  37. 37.

    Javelle, M., Marco, C. F. & Timmermans, M. In situ hybridization for the precise localization of transcripts in plants. J. Vis. Exp. 57, e3328 (2011).

  38. 38.

    Sessions, A. Immunohistochemistry on sections of plant tissues using alkaline-phosphatase-coupled secondary antibody. Cold Spring Harb. Protoc. https://doi.org/0.1101/pdb.prot4946 (2008).

  39. 39.

    Galbraith, D. W. et al. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051 (1983).

  40. 40.

    Doležel, J., Greilhuber, J. & Suda, J. Estimation of nuclear DNA content in plants using flow cytometry. Nat. Protoc. 2, 2233–2244 (2007).

  41. 41.

    Cousin, A., Heel, K., Cowling, W. A. & Nelson, M. N. An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants. Cytometry A 75A, 1015–1019 (2009).

  42. 42.

    Alexander, M. P. Differential staining of aborted and nonaborted pollen. Stain Technol. 44, 117–122 (1969).

  43. 43.

    Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

  44. 44.

    Kawahara, Y. et al. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice (N. Y.) 6, 4 (2013).

  45. 45.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  46. 46.

    Van der Auwera, G. A. et al. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr. Protoc. Bioinformatics 11, 11.10.1–11.10.33 (2013).

  47. 47.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods 25, 402–408 (2001).

Download references


We thank U. Vijayraghavan for providing pUN and pUGN vectors; S. Kappu, Z. Liechty and C. Santos-Medellín for advice and help with flow cytometry and sequence analysis; B. Van Bockern for rice transformations; and B. Nguyen and A. Yalda for technical assistance, including genotyping and transplantation. This research was supported by research grants from the National Science Foundation (NSF) (IOS-1547760) and the Innovative Genomics Institute to V.S., NSF grant IOS-1810468 to B.Y., National Institutes of Health grant 1S10OD010786-01to the University of California-Davis Genome Center, and by the United States Department of Agriculture Agricultural Experiment Station (project number CA-D-XXX-6973-H). R.M. acknowledges support from the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS) to the Institut Jean-Pierre Bourgin.

Reviewer information

Nature thanks T. Dresselhaus and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information


  1. Department of Plant Biology, University of California, Davis, CA, USA

    • Imtiyaz Khanday
    • , Debra Skinner
    •  & Venkatesan Sundaresan
  2. Innovative Genomics Institute, Berkeley, CA, USA

    • Imtiyaz Khanday
    •  & Venkatesan Sundaresan
  3. Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA

    • Bing Yang
  4. Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France

    • Raphael Mercier
  5. Department of Plant Sciences, University of California, Davis, CA, USA

    • Venkatesan Sundaresan


  1. Search for Imtiyaz Khanday in:

  2. Search for Debra Skinner in:

  3. Search for Bing Yang in:

  4. Search for Raphael Mercier in:

  5. Search for Venkatesan Sundaresan in:


V.S. and I.K. designed the study. I.K. performed experiments and analysed data. D.S. performed analysis of the genome sequences. B.Y. provided pENTR-sgRNA and pUbi-Cas9 vectors for genome editing. V.S. and I. K. wrote the manuscript with input from R.M.

Competing interests

The University of California-Davis has filed a patent application on haploid production (PCT/US2017/063249) and a provisional patent application on synthetic apomixis (US62/678,169) arising from this work. INRA has filed a patent application on the use of the MiMe system (EP2208790). The authors declare no other competing interests.

Corresponding author

Correspondence to Venkatesan Sundaresan.

Extended data figures and tables

  1. Extended Data Fig. 1 BBM1-induced somatic embryogenesis and auto-activation.

    a, Schematic of binary construct between T-DNA borders used for ectopic expression (BBM1-ox). b, Somatic embryo-like structures induced by BBM1 ectopic expression in rice leaves (n = 14/20 transgenic lines). Scale bar, 1 cm. Inset, magnified view of a somatic embryo; scale bar, 0.5 mm. Fourteen of the twenty transgenic plants raised showed the development of such embryo-like structures observed on adult seedlings from the fourth leaf onwards. c, Confirmation by RT–PCR of ectopic BBM1 expression in leaf tissues of transgenic lines. BBM1 is not expressed in wild-type leaves (n = 2 independent replicates). d, RT–PCR of embryo marker genes to confirm the embryo identity of somatic embryos induced by BBM1 overexpression. OsH1, O. sativa HOMEOBOX1; LEC1, LEAFY COTYLEDON1 (n = 2 independent biological replicates). e, Schematic of plasmid construct for DEX-inducible BBM1–GR expression system. f, Schematic showing primer combinations to distinguish between endogenous BBM1 and BBM1-GR fusion transcripts. g, RT–qPCR for fold changes in BBM1-GR fusion transcript in samples treated for 24 h with the indicated reagents, showing essentially no differences between treatments. n = 2 independent biological replicates (see Methods), data are mean ± s.e.m. and each data point represents the average fold change from three replicates. h, Autoactivation of BBM1 in samples treated with DEX for 24 h, detected by RT–qPCR. n = 2 independent biological replicates (see Methods), data are mean ± s.e.m. and each data point represents the average fold change (measured as log2(change in expression)) from three replicates.

  2. Extended Data Fig. 2 BBM1 expression in zygotes and gametes.

    a, Five SNPs sequenced after RT–PCR amplification (red arrows), showing expression only from the male allele in hybrid (J, japonica; I, indica) 2.5 HAP zygotes (n = 2 biological replicates). b, Schematic of the BBM1-GFP binary construct. c, Immunohistochemistry showing expression from both male and female BBM1 alleles in isogenic 6.5 HAP zygote nuclei (n = 20), as compared to male-specific expression at 2.5 HAP (Fig. 1a). Scale bars, 25 µm. d, Holistic view of a 6.5 HAP embryo sac showing BBM1–GFP expression in the zygote nucleus (left), while in the same embryo sac expression is not detected in the dividing endosperm (right). zg, zygote. n = 20. Scale bar 100 µm. e, BBM1–GFP expression in globular-stage rice embryos (white arrowhead, n = 30). Differential interference contrast image (left); fluorescence image (right panel). Scale bars, 200 µm. f, RT–PCR showing BBM1 expression in sperm cells; however, the transcript is not detected in egg cells (n = 2 independent biological replicates). Primers used for detecting BBM1 transcript span an intron (see Methods). g, BBM1–GFP expression in sperm cells (white arrowhead points to sperm nuclei, n = 20). Differential interference contrast image (left) and fluorescent image (right) of a germinating pollen grain showing BBM1–GFP expression in the two sperm cell nuclei.

  3. Extended Data Fig. 3 Parthenogenesis induction by expression of BBM1 in the egg cell.

    a, Schematic showing wild-type expression pattern of BBM1. b, Sketch of T-DNA region of the binary vector used for BBM1 expression in the egg cell. c, Schematic representation of the hypothesis that the expression of BBM1 in the egg cell can induce parthenogenesis. d, A degenerating parthenogenetic embryo (BBM1-ee) at 9 days after emasculation (red arrowhead). No endosperm development (black arrow) is observed in emasculated carpels, leading to the abortion of embryos (n = 12/98). Scale bar, 100 µm.

  4. Extended Data Fig. 4 CRISPR–Cas9 edited mutations in BBM1, BBM2 and BBM3 in rice.

    a, DNA sequences of mutations in bbm1/bbm1 bbm3/bbm3 plants. b, DNA sequences of mutations in bbm2/bbm2 bbm3/bbm3 plants. a and b were chosen as parents for crosses to generate the bbm1 bbm2 bbm3 triple homozygous mutants shown in c and d. c, Mutations in the F1 progeny plant. It is heterozygous for BBM1 and BBM2, and biallelic for BBM3. d, Mutations in the F2 progeny plant used for genetic analysis. The plant is heterozygous for BBM1 with a 1-bp deletion. The BBM2 locus has a homozygous 25-bp deletion and 1-bp substitution, and the BBM3 locus is a homozygous mutant with 1-bp insertion. e, Genotyping of non-germinating seeds (n = 8). The 1-bp deletion mutation in BBM1 results in disruption of an SphI restriction site. f, Seed lethality in bbm1 bbm2 bbm3 triple homozygous plants. Top, germinating one-week-old wild-type seeds (n = 30). Scale bars, 1 cm. A magnified view is shown on the right. Bottom, non-germinating seeds of bbm1 bbm2 bbm3 triple homozygous plants (n = 70). A zoomed-in image of a non-germinating bbm1 bbm2 bbm3 seed, one week after plating, is shown on the bottom right. No seedling emerged from the embryo site (red arrowhead). g, Additional image of a BBM1/bbm1 heterozygous bbm2/bbm2 bbm3/bbm3 homozygous 10 DAP embryo (n = 3/53) showing no organ formation, similar to triple homozygote phenotype (see Fig. 2a). Scale bar, 100 µm.

  5. Extended Data Fig. 5 Haploid induction and synthetic apomixis.

    Haploids shown are derived from BBM1-ee diploids by parthenogenesis. a, A control diploid sibling panicle with fertile florets (n = 442 plants). Scale bar, 1 cm. b, A haploid panicle with infertile florets (n = 113 plants). Scale bar, 1 cm. c, Differences in floret and floral organ sizes between haploid and control diploid. Left, BBM1-ee haploid; right, wild-type control (n = 20). Scale bars, 1 mm. d, Pollen viability in haploids as assessed by Alexander staining. Top, control wild-type anther with viable pollen (n = 10). Bottom, BBM1-ee haploid anther with non-viable pollen (n = 20). Scale bars, 0.5 mm (left) and 200 µm (right). e, f, Sexual reproduction compared with asexual reproduction through seed (synthetic apomixis). e, Schematic representation of sexual reproduction. Gametes form by meiotic recombination and division; fertilization and gamete fusion give rise to diploid progeny. f, Synthetic apomixis. MiMe omits meiosis and gives an unrecombined and unreduced (2n) egg cell. The 2n egg cell is converted parthenogenetically into a clonal embryo by BBM1-ee. The endosperm forms in both pathways by fertilization of central cell (homodiploid in wild type, tetraploid in synthetic apomicts) by a sperm cell (haploid in wild type, diploid in synthetic apomicts). The maternal:paternal genome ratio of 2:1 is maintained in the endosperm in both the pathways, ensuring normal seed development.

  6. Extended Data Fig. 6 Asexual propagation through seed in rice.

    a, Top, schematic of the CRISPR–Cas9 plasmid construct used for genome editing of the three MiMe rice genes. Bottom, schematic of genome-integrated pDD45::BBM1 in the BBM1-ee plants. b, DNA histogram of flow cytometric peak showing 4n ploidy in T1 progeny (n = 33/33 tested) of a control T0 MiMe plant. c, Left, panicle of a control T0 diploid MiMe plant with fertile seeds. Middle, a tetraploid T1 MiMe panicle, exhibiting complete infertility; that is, no seed filling, and larger flowers (note scale bars), with awns (white arrowhead). Awns are normally suppressed in most japonica rice cultivars including Kitaake. All T1 MiMe progeny (n = 139) were scored for the phenotype of complete infertility and presence of awns, including 33 plants that were additionally confirmed in b by flow cytometry. Right, panicle of an S-Apo haploid plant showing fertile seeds (n = 45). Scale bars, 2 cm. d, Wild-type and S-Apo haploid anthers, showing viable pollen (n = 15). Scale bars, 0.2 mm (top) and 100 µm (bottom). e, Comparison of panicles from wild type (left), with diploid clonal progeny (57/381) and sexual tetraploid progeny (n = 324/381) from a diploid S-Apo plant (right). The white arrowheads show awns in tetraploid. Scale bars, 2 cm. f, Size comparison of progeny seeds from control wild type, a synthetic S-Apo haploid, a control MiMe, a synthetic S-Apo diploid clone, and an infrequent (3%) filled seed produced by the sexual tetraploid progeny of an S-Apo diploid (n = 100 for each genotype). Scale bar, 2 mm. g, Comparison of seed size between control MiMe, diploid S-Apo line 1, diploid S-Apo line 5 and double-haploid S-Apo line DH2 (n = 100 for each transgenic line). No noticeable variation in seed size is observed. Scale bars, 2 mm.

  7. Extended Data Fig. 7 MiMe mutations and confirmation of clonal progeny from S-Apo plants.

    a, Sequence chromatograms at mutation sites of MiMe genes in wild-type, T0 diploid S-Apo mother plant and two diploid progeny from each of T1, T2 and T3 generations of S-Apo line 1 (n = 7). Red arrows point to mutation sites. PAIR1 and REC8 are biallelic whereas OSD1 is homozygous. b, Sequences of the T0 S-Apo mother plant and five T1 S-Apo diploid progeny at MiMe mutation sites and one heterozygous SNP in apomixis line 5 (n = 6). Red arrows show the mutation sites or SNP. All three MiMe mutations—OSD1, PAIR1 and REC8—are biallelic. All progeny across different generations in both the S-Apo lines have same mutations as the T0 mother plants, indicating absence of segregation and thus clonal propagation.

  8. Extended Data Fig. 8 Confirmation of SNPs by PCR.

    Sequence chromatograms of 11 SNPs are shown for wild-type, T0 diploid S-Apo mother plant and two diploid S-Apo progeny from each of the T1, T2 and T3 generations for line 1 (n = 7). All the 11 SNPs were found to be present in the T0 mother plant and all the progeny across different generations, confirming that there is no segregation; thus clonal propagation. The red arrows show the location of the SNP. Chr, chromosome; the numbers indicate the position on the respective chromosome.

  9. Extended Data Table 1 Functional characterization of BBM genes in rice
  10. Extended Data Table 2 Haploid induction and clonal propagation in rice

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Figure 1, which contains images of the uncropped agarose gels used in this study and Supplementary Table 1, which contains details of the 57 heterozygous SNPs identified in diploid S-Apo line#1.

  2. Reporting Summary

About this article

Publication history




Issue Date



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.